The present invention, in some embodiments thereof, relates to biotechnological methods for generation of products of metabolic pathways such as astaxanthin.
Native protein abundance in bacteria spans over five orders of magnitude. The balancing of protein expression levels is at the heart of proper functioning of natural biological systems and is often critical to metabolic engineering efforts. While the manipulation of intracellular protein levels, such as strong over and under-expression is widely used to elucidate the functions of biological systems, an ability to fine-tune the levels of many genes in parallel is a major outstanding challenge. In contrast to native biological systems, where the balancing of protein levels is selected for during evolution, the expression of a synthetic system can lead to imbalances in protein concentrations. As a result, synthetic pathways rarely function optimally when first introduced and the enzyme levels must be fine-tuned.
FIG. 15A schematically depicts the major challenges associated with imbalanced enzyme concentrations based on a two-step metabolic pathway model. First, low enzyme expression can limit the pathway flux and therefore product synthesis rate (blue region). At the other extreme, excessive expression might lead to protein burden, resulting in the depletion of cellular resources that limit growth (purple region). Finally, imbalances between enzymes producing and consuming an intermediate metabolite can result in a metabolic bottleneck and a high concentration of potentially toxic pathway intermediates (green region).
Approaches for controlling the intracellular abundance of proteins include altering the promoter K. Hammer, I. Mijakovic, P. R. Jensen, Synthetic promoter libraries—tuning of gene expression, Trends in Biotechnology 24, 53-55 (2006)] or the ribosome binding site (RBS) [H. M. Salis, E. A. Mirsky, C. A. Voigt, Automated design of synthetic ribosome binding sites to control protein expression, Nat Biotechnol 27, 946-950 (2009); H. H. Wang et al., Programming cells by multiplex genome engineering and accelerated evolution, Nature 460, 894-898 (2009)] sequences, modulating the stability of transcripts and varying the degradation rate of the mature protein.
Carotenoids, such as astaxanthin, are natural pigments that are responsible for many of the yellow, orange and red colors seen in living organisms. Carotenoids are widely distributed in nature and have, in various living systems, two main biological functions: they serve as light-harvesting pigments in photosynthesis, and they protect against photo oxidative damage.
Astaxanthin is the most expensive commercially used carotenoid compound (today's market value is greater than 3,500 $/kg). It is utilized mainly as nutritional supplement which provides pigmentation in a wide variety of aquatic animals. In the Far-East it is used also for feeding poultry to yield a typical pigmentation of chickens. It is also a desirable and effective nontoxic coloring for the food industry and is valuable in cosmetics. Recently it was reported that astaxanthin is a potent antioxidant in humans and thus is a desirable food additive.
Although astaxanthin is synthesized in a variety of bacteria, fungi and algae, the key limitation to the use of biological systems for its production is its low yield. One of the reasons for the low yield is the complexity of the astaxanthin pathway, whereby 7 genes of the pathway must be expressed in the cells for efficient expression. Fine tuning of the amount of expression of each of these genes is essential for optimizing astaxanthin expression.
Lemuth et al., [Microbial Cell Factories 10, 29, 2011] teaches expression of astaxanthin in E. coli. 
Salis et al., Nature Biotechnology Volume 27, No. 10, pages 946-950, 2009 teaches sequences of ribosome binding sites.
U.S. Patent Application No. 20120015849 teaches a method of creating DNA libraries that include an artificial promoter library and/or a modified ribosome binding site library and transforming bacterial host cells with the library to obtain a population of bacterial clones having a range of expression levels for a chromosomal gene of interest.